The present invention relates generally to generation holograms, and more particularly to the generation of x-ray holograms of biological specimens.
Recent advances in coherent x-ray source technology are making diffraction-limited holograms of microscopic structures, with corresponding high spatial resolution, a reality. A useful application of snapshot x-ray holography is the study of microscopic biological structures in the living state. X-rays offer high resolution and high contrast ratios for the important structures within living organisms, thereby rendering the staining of specimens, essential for optical and electron microscopy unnecessary if the wavelength is properly chosen. Picosecond time resolution, which would eliminate blurring due to thermal heating from deposition of incident energy and due to normal biological activity of the sample is also possible. Finally, with sufficiently high photon fluxes, such as those available from x-ray lasers, the x-ray snapshot can be accomplished with a single pulse yielding complete three-dimensional information of a sample having normal biological integrity at the moment of the snapshot.
A description of holographic techniques for imaging microscopic structures with a short-pulse, high intensity, high-quantum-energy laser is set forth in "Holography At X-Ray Wavelengths," by J. C. Solem, G. C. Baldwin, and G. F. Chapline, Proc. Int'l. Conf. on Lasers, pp. 293-305 (1981). Several important points therein will be summarized. First, Fresnel holography has the simplifying aspect of requiring but one laser beam. The subject specimen is placed in the laser beam itself, which beam also provides the reference. This technique, however, requires very high resolution recording media. That is, the minimum spacing which can be resolved is greater than twice the grain spacing of the medium. This result is independent of the wavelength of the incident radiation as long as the angles are small. At large diffraction angles and short wavelengths, the surface smoothness of the medium becomes important as well as its intrinsic graininess.
Fourier transform holography, by contrast, requires a reference source which emits spherical or convex curved waves, which interfere with the waves from the subject specimen at a recording surface. The specimen is illuminated by a plane wave source. The procedure is called Fourier transform holography because every distance from the reference source maps to unique spatial frequency at the recording surface. The maximum spatial frequency of the interference pattern can be adjusted arbitrarily by placing the object at various distances from the reference source. A shortcoming of the Fourier holography method described, supra, is that a spherical recording surface is required in order to obtain a complete cycle of intensity fringes for closely spaced features in the specimen. However, if the point spacing is less than the wavelength, a full cycle is never obtained. The physical spacing of the fringes can be made arbitrarily large by expanding the radius of the sphere. Therefore, ordinary film of arbitrarily large grain size could be employed as long as the trade-off between sensitivity and resolution was favorable.
In order to obtain the spherical reference wave for Fourier holography one must have a lens that focuses to a pinhole in the shadow plane. In the x-ray region of the electromagnetic spectrum a Fresnel zone plate is used to accomplish this. However, the hologram resolution is limited to finest spacing on such a plate, currently about 10 nm. An alternative would be to use a coherent scattering backward reflector to generate the spherical reference waves. In FIG. 6 of the Solem reference, supra, the authors show a parabolic reflector enclosed in a spherical shell recording surface. For best contrast ratio, the paraboloid would have to be approximately the same size as the object. The reference scatterer need not be a paraboloid. In principle, the hologram could be unfolded for any convex reference scatterer as long as the shape and dimensions thereof were known to within a fraction of a wavelength.
In "X-Ray Biomicroholography," by Johndale C. Solem and George F. Chapline, Opt. Eng. 23, 203 (1984), the authors state that most of the information about the fine details of the specimen appears at large scattering angles and can be degraded by recording surface roughness. The problem is mitigated by using a spherical recording surface. However, the reference scatterer will have a low scattering efficiency, as will the specimen, and the intensity of reference- and specimen-scattered waves will approximately match for highest contrast. The authors also discuss briefly the use of an x-ray photocathode and microchannel plates. However, the authors state that such devices saturate easily, have a small dynamic range, and are available only in fairly small sizes.
Accordingly, it is one object of the present invention to provide an apparatus for recording high resolution x-ray holograms.
Another object of our invention is to provide an apparatus for electrically recording high resolution x-ray holograms of biological samples using currently available electronics technology.
Yet another object of the present invention is to provide a method for obtaining a faithful reproduction of objects from detected holograms thereof.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.